Mouse liver microsomal esterases were fractionated on DEAE-cellulose after the solution of these enzymes in a solution containing 0.1 M glycyl glycine buffer, pH 7.0, and 5 × 10−4 M Lubrol W, a nonionic detergent. Elution of the enzymes from the DEAE-cellulose was accomplished by using NaCl in the glycyl glycine – Lubrol W solution. Microsomes isolated from sucrose and glycerol homogenates have three esterase peaks in common and two peaks not in common. The glycerol supernatant fraction from whole liver homogenate, which contains about 50% of the total esterase activity, and the sucrose supernatant fraction, which contains about 6% of the total esterase activity, have four common esterase peaks, one of which is different from both the microsomes isolated from glycerol and sucrose liver homogenates. Incorporation of Lubrol W in the solution that was employed for the elution of the esterases from the DEAE-cellulose was necessary to prevent extensive loss or inactivation of these enzymes. The nature of the changes induced by glycerol on liver esterases is briefly discussed.